title: The antiallergic mast cell stabilizers lodoxamide and bufrolin as the first high and equipotent agonists of human and rat GPR35.
abstract: Lack of high potency agonists has restricted analysis of the G protein-coupled receptor GPR35. Moreover, marked variation in potency and/or affinity of current ligands between human and rodent orthologs of GPR35 has limited their productive use in rodent models of physiology. Based on the reported modest potency of the antiasthma and antiallergic ligands cromolyn disodium and nedocromil sodium, we identified the related compounds lodoxamide and bufrolin as high potency agonists of human GPR35. Unlike previously identified high potency agonists that are highly selective for human GPR35, both lodoxamide and bufrolin displayed equivalent potency at rat GPR35. Further synthetic antiallergic ligands, either sharing features of the standard surrogate agonist zaprinast, or with lodoxamide and bufrolin, were also shown to display agonism at either human or rat GPR35. Because both lodoxamide and bufrolin are symmetric di-acids, their potential mode of binding was explored via mutagenesis based on swapping between the rat and human ortholog nonconserved arginine residues within proximity of a key conserved arginine at position 3.36. Computational modeling and ligand docking predicted the contributions of different arginine residues, other than at 3.36, in human GPR35 for these two ligands and were consistent with selective loss of potency of either bufrolin or lodoxamide at distinct arginine mutants. The computational models also suggested that bufrolin and lodoxamide would display reduced potency at a low-frequency human GPR35 single nucleotide polymorphism. This prediction was confirmed experimentally.
authors.affiliation: Molecular Pharmacology Group, Institute of Molecular, Cell, and Systems Biology (A.E.M., G.C., L.J., J.E.M., B.D.H., G.M.) and Institute of Cardiovascular and Medical Sciences, (J.E.M., S.A.N.), College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom; Laboratory of Computational Medicine, Biostatistics Unit, Faculty of Medicine, Autonomous University of Barcelona, Bellaterra, Spain (G.C.); and Novartis Institutes for Biomedical Research, Horsham, United Kingdom (T.C.K., L.F., R.M., S.J.C.).
authors.forename: Amanda E
authors.initials: AE
authors.lastname: MacKenzie
authors.forename: Gianluigi
authors.initials: G
authors.lastname: Caltabiano
authors.forename: Toby C
authors.initials: TC
authors.lastname: Kent
authors.forename: Laura
authors.initials: L
authors.lastname: Jenkins
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: McCallum
authors.forename: Brian D
authors.initials: BD
authors.lastname: Hudson
authors.forename: Stuart A
authors.initials: SA
authors.lastname: Nicklin
authors.forename: Lindsay
authors.initials: L
authors.lastname: Fawcett
authors.forename: Rachel
authors.initials: R
authors.lastname: Markwick
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Charlton
authors.forename: Graeme
authors.initials: G
authors.lastname: Milligan
mesh_terms: D000818:Animals; D018926:Anti-Allergic Agents / Q000494:pharmacology*; D002460:Cell Line; D003198:Computer Simulation; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D008407:Mast Cells / Q000187:drug effects* / Q000502:physiology; D062105:Molecular Docking Simulation; D009154:Mutation; D010072:Oxamic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D010618:Phenanthrolines / Q000494:pharmacology*; D020641:Polymorphism, Single Nucleotide; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000819:agonists* / Q000235:genetics
chemical_list: D018926:Anti-Allergic Agents; C111379:GPR35 protein, human; C513985:GPR35 protein, rat; D010618:Phenanthrolines; D043562:Receptors, G-Protein-Coupled; C100241:bufrolin; D010072:Oxamic Acid; C021702:lodoxamide ethyl